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Katja Galea, Borut trukelj, Sao Bavec, Vito Turk, Brigita Lenarcic

Cloning and Expression of Functional Equistatin

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(s+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 381, 01/2000
Pages: 85 - 88

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